1. Field of Invention
The present invention relates to a diagnostic composition and method for the detection of an infection by the nematode Trichinella spp in a body fluid sample from animals or humans, preferably in serum or meat juice of pigs or other susceptible organisms and to diagnostic kits that can be used for monitoring and surveillance for Trichinella infections or for testing of individual carcasses for food safety.
2. Description of Related Art
Trichinella spp. is a group of nematodes that can occur worldwide. There are eight different species; the species of main importance in Europe are Trichinella spiralis, Trichinella britovi, Trichinella pseudospiralis, and Trichinella nativa. Trichinella spp. can infect a wide variety of species including humans, pigs, rats, bears, horses and birds.
The parasitic nematode Trichinella undergoes a distinct live cycle. Larvae are ingested by eating raw or undercooked meat. They are released from nurse cells in the stomach from where they enter the small intestine, where adults mature. Females shed newborn larvae that enter the lymph, reach the venous blood and settle in voluntary muscles, especially those of the diaphragm, tongue and masseters. Larvae mature and form nurse cell-larvae complexes that calcify.
Trichinella infection in production animals is important because of the risk for humans to contract trichinellosis following consumption of raw or undercooked meat from infected animals. Human trichinellosis is a serious disease that can cause much suffering and may result in death.
There are three different diagnostic methods to detect Trichinella spp. in animals.
Two methods are based on the detection of the parasite; the third method is based on the detection of antibodies against this nematode.
The first method called “Trichinoscopy” or the compressorium method is a direct detection method in which tissue is compressed between two glass plates and studied microscopically. This method is rather insensitive and labour-intense. More important, non-encapsulating Trichinella species such as T. pseudospiralis may not be detected with this method.
The second method called “digestion method” is a more sensitive direct detection method in which muscle tissue surrounding the larvae is artificially digested to release the larvae. Subsequently, the number of larvae can be determined microscopically. The detection limit by artificial digestion of 1 g tissue is 3-5 larvae per gram.
The third method is an indirect detection method using serology to detect antibodies against Trichinella spp. As antigens the excretory/secretory (E/S) antigens can be used. Although this method is currently not recommended for meat inspection or food safety programs, it is an important tool for surveillance programs and epidemiological investigations in animal populations. Serology can be based on blood serum or meat juice. This method has a limit of detection of 0.01 larvae per gram tissue.
For the purpose of ensuring food safety, all pig carcasses must be tested as part of the post-mortem examination. Carcasses may be cut up in a maximum of six parts, or be cut up in a cutting plant adjacent to the slaughterhouse before the results of the Trichinella tests are available, but further processing can only take place after the Trichinella tests have shown negative results.
According to EU Regulation Trichinoscopy will no longer be permitted as a standard method of examination, although it may be used as a transitional measure during a period of maximum four years following the date of application of the new EU Regulation.
The prescribed method for Trichinella examinations will be the digestion method with the magnetic stirrer method for pooled sample digestion. Equivalent methods are the mechanically assisted (Stomacher) pooled sample digestion method using ‘sedimentation technique’, the mechanically assisted (Stomacher) pooled sample digestion method using ‘on filter isolation technique’, and the automatic digestion method (Trichomatic 35) for pooled samples of up to 35 gram.
Currently used Trichinella digestion tests have a number of disadvantages.
All digestion methods are labour intensive as they require manual inspection of the filtered digestion mixture by microscopy performed by an experienced investigator. No routine training of investigators or testing of proficiency panels to assure validity of the inspection procedure is provided by current EU regulation. As a rule the investigators performing the digestion method know positive test results only from their initial training.
Finally currently approved test methods do not allow tracking of the samples and documentation of the results.
At present as stated above serological methods are considered useful for monitoring purposes but are not considered suitable for the detection of Trichinella infection in individual animals intended for human consumption. In most cases problems in known serological methods result from the diagnostic antigens used. The presently used crude antigens or E/S antigens include antigenic components that may react non-specifically and thus produce false positive results. Additionally the diagnostic window and selectivity of known serological tests using e.g. E/S antigens is considered to be too small. Finally, known serological methods require special technical and personal equipment which at present is not available in slaughterhouses.
When comparing both systems it appears that on principle serological tests have a number of advantages over digestion methods and that if improved they could be an interesting alternative also in routine testing.
Some of the disadvantages mentioned above can be met by using different antigens. In this context Ting-Xian et al. e.g. describe (Chin. J. Parasitol. Dis.; June 2005, No. 3; 143) the use of T668 antigen as diagnostic antigen in immunoassays. However, also here the problems regarding the narrow diagnostic window remain.